Modern healthcare relies extensively on a range of chemical and biochemical analytical tests on a variety of body fluids to enable diagnosis, therapy and management of disease. Medical and technological advances have considerably expanded the scope of diagnostic testing over the past few decades. Moreover, an increasing understanding of the human body, together with the emergence of technologies, such as microsystems and nanotechnology, are expected to have a profound impact on diagnostic technology.
Increasingly, diagnostic tests in hospitals are carried out at the point-of-care (PoC), in particular, in situations where a rapid response is a prime consideration and therapeutic decisions have to be made quickly. Despite recent advances in PoC testing, several compelling needs remain unmet. For example, the detection of small molecules in biological samples is often very challenging, especially when no suitable receptor (e.g. enzyme, antibody, aptamer) with an appropriate specificity exists. The challenge is even greater when the molecule is lipophilic and a large proportion of the analyte is unavailable for analysis due to its association with hydrophobic components of the sample matrix, such as cells, lipids and proteins.
The detection of small molecules in complex media (e.g. blood, plasma, saliva, urine, waste water and their extracts) is often difficult due to the association of the analyte with components of the sample matrix (e.g. plasma proteins and lipid membranes). The free (unbound) molecule concentration (which can be in the picomolar range) is often below the sensitivity limits of the most commonly used measurement techniques (e.g. electrochemical, optical). For this reason, state of the art methods for small molecule detection in complex media often involve intensive sample preparation, such as dilution/extraction of the sample into an organic solvent, centrifugation, evaporation and analysis by high pressure liquid chromatography (HPLC). Depending on the specific characteristics of the analyte molecule, post-HPLC column detection of the eluted compound is performed using electrochemical or optical (absorption spectroscopy or fluorescence) methods, such as disclosed in G. F. Plummer, “Improved method for the determination of propofol in blood by high-performance liquid chromatography with fluorescence detection,” Journal of Chromatography, vol. 421, 1987, p. 171 and in R. A. Uebel et al., “Electrochemical determination of 2,6-diisopropylphenol after high-performance liquid chromatography of extracts from serum,” Journal of Chromatography, vol. 526, March 1990, pp. 293-5.
The complex and time-consuming nature of HPLC assays for small molecules in complex samples means that they are routinely performed by a very small number of specialist laboratories; for this reason the utility of these assays is rather limited. For example, for many drugs, such as propofol, there is a clear need to develop alternative, miniaturised assays. This would enable measurement and clinical intervention close to real time and at the point-of-care (PoC).
A method for detecting and measuring propofol in complex media has been described by McGaughran et al., “Rapid measurement of blood propofol levels: A proof of concept study,” Journal of Clinical Monitoring and Computing, vol. 20, 2006, pp. 381-381. The disclosed method comprises solid phase extraction (SPE) of a diluted whole blood sample, followed by reaction with a phenol-specific (Gibbs) reagent, namely 2,6 dichloroquinone-4-chloroimide, to produce a strongly coloured indophenol product and detection of this same product by absorption spectroscopy.
The Gibbs/indophenol method has been successfully utilised for the detection of propofol. Here, specificity is achieved by the combination of the SPE step (specific for hydrophobic analytes), and the Gibbs reaction (specific for para-unsubstituted phenols, such as propofol, as for instance has been described by D. Svobodová at al., “Colour reaction of phenols with the Gibbs reagent. The reaction mechanism and decomposition and stabilisation of the reagent,” Microchimica Acta, vol. 67, May. 1977, pp. 251-264, and by H. D. Gibbs, “Phenol tests III. The indophenol test,” Journal of Biological Chemistry vol. 72, 1927, pp. 649-664.
The overall process for the assay is described in FIG. 1, while the dominant reaction scheme for propofol conversion to the indophenol is shown in FIG. 2. As shown in FIG. 1, the process begins at step 10 in which a whole blood sample is diluted with distilled water in a ratio of 1:2. In step 20, the dilution product is injected over a SPE column, followed by a washing step 30 in which the SPE column is washed with a mixture of water and 50% methanol to remove weakly bound impurities. Next, propofol is extracted from the SPE column by elution with acetonitrile as shown in step 40, after which the Gibbs reaction is performed involving propofol as a reagent to produce a coloured indophenol product in step 50. To determine the propofol concentration in the original blood sample, the associated coloured indophenol concentration is determined in step 60 using visible absorption spectroscopy. It is noted that the Gibbs reaction is specific for all para-unsubstituted phenols including propofol. The potential for interference in the Gibbs reaction from other phenols is reduced by the SPE extraction step.
At sufficiently high pH and in the presence of a primary or secondary alcohol, the Gibbs reagent (A) is rapidly converted to an active form (B) which in turn reacts with propofol (C) to produce a coloured indophenol product (D), as shown in FIG. 2 and described in detail by D. Svobodová et al., “The colour reaction of phenols with the Gibbs reagent,” Microchimica Acta, vol. 70, 1978, pp. 197-211. At a pH greater than 9.5, the rate of conversion of the Gibbs reagent (A) to the activated form (B) is much greater than the rate of reaction between propofol and the activated Gibbs reagent. In this case, the formation of the indophenol product from the reaction between the activated Gibbs reagent and propofol is the rate limiting step. Therefore, at high pH and when the concentration of the Gibbs reagent is in excess relative to propofol, the concentration of the indophenol product at equilibrium and the initial concentration of propofol in the sample are proportional to each other. Hence, an equilibrium measurement of the absorbance peak of the indophenol product at 595 nm, which by the Beer-Lambert law is directly proportional to the indophenol concentration, will give the initial propofol concentration in the sample before the reaction.
Testing of a device which utilises SPE and the Gibbs/indophenol reaction for propofol measurement has revealed excellent precision, linearity and accuracy for propofol concentrations down to 1 μg/ml in whole blood, as disclosed by McGaughran et al., “Rapid measurement of blood propofol levels: A proof of concept study,” Journal of Clinical Monitoring and Computing, vol. 20, 2006, pp. 381-381. This limit of detection makes the device especially suitable for propofol measurements during surgical operations: during surgery, patients are usually administered sufficient propofol to ensure that the average blood propofol concentration is well above 2 μg/ml. For example, Schafer et al., “Pharmacokinetics and pharmacodynamics of propofol infusions during general anesthesia,” Anesthesiology, vol. 69, September 1988, pp. 348-356, reported that patients required an average blood propofol concentration of 4.05+/−1.01 μg/ml for major surgery and 2.97+/−1.07 μg/ml for non-major surgery. Blood propofol concentrations at which 50% of patients (EC50) were awake and oriented after surgery were 1.07 and 0.95 μg/ml, respectively. This method and apparatus is more suited to use in these settings than the HPLC-based techniques, since the sample preparation is straight-forward, requiring only a simple blood sample dilution before introduction to the device. Furthermore, typical measurement times are much faster at approximately 3 to 5 minutes. Moreover, the instrument has a much smaller footprint and lower complexity than the equivalent HPLC device.
In settings where propofol is used for sedation, such as the intensive care unit (ICU), the propofol concentrations in whole blood are typically in the region of 0.25 to 2 μg/ml, as disclosed by J. Barr et al., “Propofol dosing regimens for ICU sedation based upon an integrated pharmacokinetic-pharmacodynamic model,” Anesthesiology, vol. 95, 2001, p. 324. There is therefore a need to extend the lower limit of detection and measurement for the propofol assay described above. However, the optical measurement of propofol in whole blood below 1 μg/ml is limited by the presence of species in the blood sample that bind to and co-elute from the SPE column and absorb in the region of the indophenol signal.
In addition, insoluble aggregates can also be present and can scatter the light, thereby reducing the measured intensity at the detector. These species contribute to the measured absorbance spectrum in the region of the indophenol signal at 595 nm, causing an offset in the measured absorbance at this wavelength and limiting the ability of the instrument to measure accurately the propofol concentration in blood samples containing less than 1 μg/ml of propofol. As this non-propofol signal varies for different blood samples, this offset due to interfering species cannot be corrected for by applying, for example, correlation factors to the data. It is theoretically possible to correct for the extra absorbance signal using knowledge of a larger part of the absorbance spectrum (depending on the nature of the interfering species present). However, it would necessitate the use of expensive spectrometers to measure the absorbance signal either side of the absorbance peak of interest and interpolating the signal in order to subtract the background contribution to the peak. This, in turn, will increase the cost and complexity of the device.